An ancillary development stemming from researchers' ability to produce and amplify recombinant proteins, and the genes from which they are encoded, is the high-throughput microarray. While initial applications of high-throughput screening focused on genomic arrays (Schena et al., “Quantitative Monitoring of Gene Expression Patterns With a Complementary DNA Microarray,” Science 270:467-470 (1995), Lipshutz et al., “High density Synthetic Oligonucleotide Arrays,” Nat. Genet. 21:20-24 (1999)), the protein microarray has found a variety of significant uses as well. For example, proteome profiling via protein microarrays has unveiled a myriad of novel interactions (MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination,” Science 289:1760-1763 (2000), Michaud et al., “Analyzing Antibody Specificity with Whole Proteome Microarrays,” Nat. Biotech. 21:1509-1512 (2003) and Chan et al., “Protein Microarrays for Multiplexed Analysis of Signal Transduction Pathways,” Nat. Med. 10:1390-1396 (2004)). Protein microarrays have been used to discover antigenic proteins and monitor human immunological responses to them (Davies et al., “Profiling the Humoral Immune Response to Infection by Using Proteome Microarrays: High-Throughput Vaccine and Diagnostic Antigen Discovery,” Proc. Natl. Acad. Sci. U.S.A. 102:547-552 (2005); Li et al., “Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine,” Infect. Immun. 73:3734-3739 (2005); and Qiu et al., “Antibody Responses to Individual Proteins of SARS Coronavirus and Their Neutralization Activities,” Microbes Infect. 7:882-889 (2005)). This tactic has not been used previously for immobilization of multiple antibodies or antibody binding fragments for serological detection of Streptococcus pneumoniae. Moreover, in each of these reports, detection was achieved using labeled reagents.
Previous reports (Horner et al., “A Proteomic Biosensor for Enteropathogenic E. coli,” Biosen. Bioelect. 21:1659-1663 (2006) and Mace et al., “Theoretical and Experimental Analysis of Arrayed Imaging Reflectometry as a Sensitive Proteomics Technique,” Anal. Chem. 78:5578-5583 (2006)) describe arrayed imaging reflectometry detection of two interacting proteins, but have been unable to provide full serotype-level identification. Likewise, Chan et al., “Identification of Gram Negative Bacteria Using Nanoscale Silicon Microcavities,” J. Am. Chem. Soc. 123:11797-11798 (2001), acknowledges that a liposaccharide sensor is not able to discriminate between different types of Gram-(−) bacteria. There has been some doubt as to whether, e.g., arrayed imaging reflectometry of capsular polysaccharides would be defeated by non-specific binding.
Streptococcus pneumoniae continues to be an exceptionally important human pathogen. Of the estimated 1.3 million global cases of childhood pneumonia in 2011 leading to death, 18.3% (237,900) were caused by S. pneumoniae (Fischer et al., “Global Burden of Childhood Pneumonia and Diarrhea,” Lancet 381:1405-1416 (2013)). Pneumococcal pneumonia is also a significant problem in adults (Said et al., “Estimating the Burden of Pneumococcal Pneumonia among Adults: A Systematic Review and Meta-analysis of Diagnostic Techniques,” PLoS One 8(4):e60273 (2013)). As more than 90 distinct serotypes of S. pneumoniae exist, the organism presents a substantial challenge to serology.
In addition to the traditional Quellung reaction method (Lund, “Laboratory Diagnosis of Pneumococcus Infections,” Bull. World Health Organ. 23:5-13 (1960)), recent advances in S. pneumoniae detection have included the development of competition bead-based immunoassays (Yu et al., “A Rapid Pneumococcal Serotyping System Based on Monoclonal Antibodies and PCR,” J. Med. Microb. 57:171-178 (2008)). While an important advance, such assays still require complex instrumentation and an equally complex workflow. Additionally, as with all sandwich immunoassays, increasing the number of serotypes covered by the assay is complicated by the need to re-qualify each component of the assay for cross-reactivity. PCR-based methods are widely used and popular, but these are indirect, complex, expensive, and there are recognized limitations to their strain coverage (Menezes et al., “Update of Pneumococcal ‘PCR-Serotyping’ for Detection of a Commonly Occurring Type 19F wzy Variant in Brazil,” J. Clin. Microbiol. Doi:10.112/JCM.00743-13 (2013)).
Given the current state of the art and importance to human health, new serological methods for pneumococcus are clearly needed.
It would be desirable to provide an array of immobilized antibodies or antibody binding fragments that can be used to distinguish between different serotypes of Streptococcus pneumoniae. 